Thrombocytopenia is the disease of platelet deficiency caused by anticancer therapy, bone marrow graft and so on. In the process of anticancer therapy or bone marrow graft, megakaryocyte colony forming cells, the platelet precursor cells in bone marrow, are disrupted, and this leads to platelet deficiency. The thrombocytopenia patient is subject to bleeding in response to a light trauma, and more serious patient becomes bleeding without trauma. Bleeding is often fatal in this case since the blood is not stanched at all.
The current therapy for thrombocytopenia is nothing but the platelet transfusion. However, several problems and side effects are associated with this therapy, such as insufficient donors, transfusion-meditated infection with e.g. HIV (human immunodeficiency virus) and hepatitis viruses, the elicitation of immune response, and so on.
Platelet is a component of blood, originated from megakaryocyte precursor cells, and plays a role in the suppression of bleeding. Thrombopoietin (hereafter, referred to as “TPO”), a glycoprotein synthesized and secreted in liver or kidney, regulates the platelet level in blood. TPO accelerates the proliferation and differentiation of the megakaryocyte precursor cells, which is followed by the platelet production (Lok et al., Nature, 369: 565–568, 1994; De savage et al., Nature, 369: 533–568, 1994).
Since a gene encoding TPO was isolated first from human in 1994 (Lok et al., Nature, 369: 565–568, 1994; De savage et al., Nature, 369: 533–568, 1994; Miyazaki et al., Experimental hematol., 22: 838, 1994; WO 95/18858), clinical approaches for thrombocytopenia have been based on the function of human TPO (hereinafter, referred to as “hTPO”), that is, the regulation of the platelet level.
Three different approaches are proceeded in order to improve the activity of native hTPO.
Glycoprotein hTPO is expressed in cells as an inactive precursor comprising 353 amino acids, and the cleavage of signal peptide (21 amino acids) leads to the secretion of active hTPO protein (332 amino acids) out of the cells. The amino acid sequence of hTPO is divided into two regions. The N-terminal region comprising 151 amino acids contains catalytic site, and shows high similarity to that of erythropoietin (; EPO) The other region, C-terminal region is presumed to have a key role in the extracellular secretion and in vivo stability of hTPO.
The first method for modifying native hTPO relates to the deletion of the C-terminal region or the addition of new amino acids to the deleted hTPO.
In support of this approach, Amgen INC. developed various hTPO derivatives such as hTPO151 (consisting of amino acids 1–151), hTPO174 (consisting of amino acids 1–174) and the hTPO163 supplemented with methionine-lysine in its N-terminal. However, these derivatives proved to show lower hTPO activity in vivo than native hTPO, although their activities were maintained in vitro (WO 95/26746, WO 95/25498).
In addition, Genentech INC. prepared from E. coli a recombinant hTPO153 derivative having an N-terminal methionine (WO 95/18858). Kirin produced diverse hTPO derivatives with C-terminal deletion and hTPO163 derivatives with substitution, deletion, or insertion at a specific amino acid residue (WO 95/21920). Other hTPO derivatives with C-terminal deletion were provided by Zymogenetics INC. (WO 95/21920; WO 96/17062) and G. D. Searl (WO 96/23888). These derivatives, however, failed to show higher activity of platelet production in vivo than native hTPO.
The second method is associated with the conjugation of polyethyleneglycol (; PEG) with hTPO fragment, which is exampled by hTPO163-PEG of Amgen INC. (WO 95/26746).
The derivatives according to this method, however, have critical handicaps such as poor stability and safety, since they do not contain C-terminal region that is important for the stability of hTPO and since immune response may be elicited by the shift of their folding structures. Moreover, the qualities of products may be uneven because PEG is not so conjugated at a uniform proportion.
The third method exploits the glycosylation of hTPO, which may increase the hTPO activity.
Amgen INC. performed a mutagenesis where a specific nucleotide in cDNA encoding hTPO was substituted to bear amino acid sequence “Asn-X-Ser/Thr” (where X is any amino acid but proline). The mutated gene was used to prepare hTPO derivatives with C-terminal deletion, which comprised 174 amino acids and into which one or more N-linked glycosylation sites are produced (WO 96/25498).
Korea Research Institute of Biology and Biotechnology (KRIBB) produced a hTPO derivative where one sugar chain is incorporated into the intact native hTPO (Park et al., J. Biol. Chem., 273: 256–261, 1998), distinctive from the Amgen's partial hTPO derivatives.
However, all these derivatives did not show significantly higher levels of hTPO activity.
As described above, although various strategies have been employed to develop hTPO derivatives with enhanced biological activity, all failed to obtain the derivatives with higher in vivo hTPO activities than native hTPO.
Generally, numerous proteins exist as proteins adorned by oligosaccharide chains in specific position, i.e. glycoproteins. Two types of glycosylation have been found. In O-linked glycosylation, sugar chain is attached to the hydroxyl group of Ser/Thr residue in the glycoprotein. In N-linked glycosylation, sugar chain is attached to the amide group of “Asn-X-Ser/Thr” (X is any amino acid but proline).
The sugar chain in a glycoprotein exert various effects on the physical, chemical and biological properties such as protein stability and secretion, especially on the biological activity in vivo and pharmacokinetic properties (Jenkins et al., Nature Biotechnological., 14: 975–981, 1996; Liu et al., Act. TIBTECH., 10: 114–120, 1992).
These effects are exemplified by human interferon-γ and glucose transport protein, where amino acid substitution at proper glycosylation site gave rise to the striking decrease in the hTPO activity, suggesting that N-linked sugar chain may have significant effects on the activity of the glycoprotein (Sareneva et al., Biochem. J. 303: 831–840, 1994; Asano et al., FEBS, 324: 258–261, 1993).
However, the introduction of additional sugar chains is not always accompanied with an increase in the catalytic activity of the glycoprotein, as described in the precedent art of Amgen INC. and KRIBB (WO 96/25498; Park et al., J. Biol. Chem., 273: 256–261, 1993). Although additional sugar chains were introduced into these hTPO derivatives, the biological activities of the glycoproteins were rather reduced when compared with native hTPO. According to this observation, it is not the number of sugar chains but the specific glycosylation site that is crucial for elevating its catalytic activity.
We, the inventors of this invention, have prepared various hTPO derivatives and examined their activities. This invention is performed by disclosing that several hTPO derivatives such as derivative wherein Asn is substituted for Arg164; derivative wherein Asn is substituted for Thr193; derivative wherein Asn is substituted for Pro157 and Arg164; and derivative wherein Asn is substituted for Leu108, Arg117 and Arg164 produce the remarkably higher levels of platelets than native hTPO does, which is not ever observed in the current hTPO derivatives.